In our genome-wide screens for small RNAs, we found that a number of short RNAs actually encode small proteins. The correct annotation of the smallest proteins is one of the biggest challenges of genome annotation, and perhaps more importantly, few annotated short ORFs have been confirmed to correspond to synthesized proteins. Although these proteins have largely been missed, the few small proteins that have been studied in detail in bacterial and mammalian cells have been shown to have important functions in signaling and in cellular defenses. Thus we established a project to identify and characterize E. coli proteins of less than 50 amino acids. Identification of small proteins We used sequence conservation and ribosome binding site models to predict genes encoding small proteins, defined as having 16-50 amino acids, in the intergenic regions of the Escherichia coli genome. We tested expression of these predicted as well as previously annotated small proteins by integrating the sequential peptide affinity tag directly upstream of the stop codon on the chromosome and assaying for synthesis using immunoblot assays. This approach confirmed that 20 previously annotated and 18 newly discovered proteins of 16-50 amino acids are synthesized. Remarkably more than half of the newly discovered proteins are predicted to be single transmembrane proteins, nine of which we show co-fractionate with cell membranes. Characterization of small proteins We are employing many of the approaches the group has used in characterizing the functions of small regulatory RNAs to elucidate the functions of the small proteins. Systematic assays for the accumulation of tagged versions of the proteins have shown that many small proteins accumulate under specific growth conditions or after exposure to stress. We also generated and screened bar-coded null mutants and identified small proteins required for resistance to cell envelope stress and acid shock. The attached sequential peptide affinity tag is now being exploited to purify the small protein and identify co-purifying complexes. The combination of these approaches will give insights into when, where and how the small proteins are acting.